Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

Oxidized and reduced Azotobacter vinelandii ferredoxin I at 1.4 A resolution: conformational change of surface residues without significant change in the [3Fe-4S]+/0 cluster.

The refined structure of reduced Azotobacter vinelandii 7Fe ferredoxin FdI at 100 K and 1.4 A resolution is reported, permitting comparison of [3Fe-4S]+ and [3Fe-4S]0 clusters in the same protein at near atomic resolution. The reduced state of the [3Fe-4S]0 cluster is established by single-crystal EPR following data collection. Redundant structures are refined to establish the reproducibility and accuracy of the results for both oxidation states. The structure of the [4Fe-4S]2+ cluster in four independently determined FdI structures is the same within the range of derived standard uncertainties, providing an internal control on the experimental methods and the refinement results. The structures of the [3Fe-4S]+ and [3Fe-4S]0 clusters are also the same within experimental error, indicating that the protein may be enforcing an entatic state upon this cluster, facilitating electron-transfer reactions. The structure of the FdI [3Fe-4S]0 cluster allows direct comparison with the structure of a well-characterized [Fe3S4]0 synthetic analogue compound. The [3Fe-4S]0 cluster displays significant distortions with respect to the [Fe3S4]0 analogue, further suggesting that the observed [3Fe-4S]+/0 geometry in FdI may represent an entatic state. Comparison of oxidized and reduced FdI reveals conformational changes at the protein surface in response to reduction of the [3Fe-4S]+/0 cluster. The carboxyl group of Asp15 rotates approximately 90 degrees, Lys84, a residue hydrogen bonded to Asp15, adopts a single conformation, and additional H2O molecules become ordered. These structural changes imply a mechanism for H+ transfer to the [3Fe-4S]0 cluster in agreement with electrochemical and spectroscopic results.     

Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of the metal clusters in the DeltanifB and DeltanifH mofe proteins of nitrogenase from Azotobacter vinelandii

Deletion of nifB results in the formation of a variant nitrogenase MoFe protein (DeltanifB MoFe protein) that appears to contain two normal [8Fe-7S] P clusters. This protein can be reactivated to form the holo MoFe protein upon addition of isolated FeMo cofactor. In contrast, deletion of nifH results in a variant protein (DeltanifH MoFe protein) that appears to contain FeS clusters different from the normal P cluster, presumably representing precursors of the normal P cluster. The DeltanifH MoFe protein is not reconstituted to the holo MoFe protein with isolated FeMo cofactor. The EPR and EXAFS spectroscopic properties of FeS clusters in the DeltanifH MoFe protein clearly differ from those of the normal P cluster found in the DeltanifB MoFe protein and suggest the presence of [4Fe-4S]-like clusters. To further characterize the metal cluster structures in the DeltanifH MoFe protein, a variable-temperature, variable-field magnetic circular dichroism (VTVH-MCD) spectroscopic study has been undertaken on both the DeltanifB MoFe protein and the DeltanifH MoFe protein in both the dithionite-reduced and oxidized states. This study clearly shows that each half of the dithionite-reduced DeltanifH MoFe protein contains a [4Fe-4S]+ cluster paired with a diamagnetic [4Fe-4S]-like cluster. Upon oxidation, the VTVH-MCD spectrum of the DeltanifH MoFe protein reveals a paramagnetic, albeit EPR-silent system, suggesting an integer spin state. These results suggest that the DeltanifH MoFe protein contains a pair of neighboring, unusual [4Fe-4S]-like clusters, which are paramagnetic in their oxidized state.     

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum

The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.     

Crystallographic analysis of two site-directed mutants of Azotobacter vinelandii ferredoxin

The crystal structure of the C24A mutant of Azotobacter vinelandii 7Fe ferredoxin (FdI) has been solved and refined at 2.0-A resolution. The structure is isomorphous to native FdI except at the site of mutation where A24 moves toward the [4Fe-4S] cluster. In spite of this inefficient packing results: three of five van der Waals contacts from the S gamma of C24 in native FdI are lost and the remaining two become longer. Consequently, the [4Fe-4S] cluster is either disordered or has a higher temperature factor (B factor) compared to the rest of the C24A FdI molecule. In addition, the entire C24A FdI structure has a higher overall B factor than native FdI. Therefore, in comparison to native FdI, the C24A mutant is isomorphous but exhibits large differences in B factor, especially at the [4Fe-4S] cluster. In contrast, the C20A FdI structure (Martin, A. G., Burgess, B. K., Stout, C. D., Cash, V. L., Dean, D. R., Jensen, G. M., and Stephens, P. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 598-602), which contains large structural rearrangements in the vicinity of the [4Fe-4S] cluster, exhibits essentially no change in B factor. The conformational change observed at residue 24 is similar in both C24A and C20A FdI structures. The solvent accessibility of the Fe atoms in the [3Fe-4S] and [4Fe-4S] clusters is similar in C24A, C20A, and native FdI.     

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I. Changes in [4Fe-4S] cluster reduction potential and reactivity.

We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.     

Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.     

Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas.

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.     

The three-iron cluster in a ferredoxin from Desulphovibrio gigas. A low-temperature magnetic circular dichroism study

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.     

A structural basis of Equisetum arvense ferredoxin isoform II producing an alternative electron transfer with ferredoxin-NADP+ reductase

We have determined the crystal structure, at 1.2-A resolution, of Equisetum arvense ferredoxin isoform II (FdII), which lacks residues equivalent to Arg(39) and Glu(28) highly conserved among other ferredoxins (Fds). In other Fds these residues form an intramolecular salt bridge crucial for stabilization of the [2Fe-2S] cluster, which is disrupted upon complex formation with Fd-NADP(+) oxidoreductase (FNR) to form two intermolecular salt bridges. The overall structure of FdII resembles the known backbone structures of E. arvense isoform I (FdI) and other plant-type Fds. Dramatically, in the FdII structure a unique, alternative salt bridge is formed between Arg(22) and Glu(58). This results in a different relative orientation of the alpha-helix formed by Leu(23)-Glu(29) and eliminates the possibility of forming three of the five intermolecular salt bridges identified on formation of a complex between maize FdI and maize FNR. Mutation of FdII, informed by structural differences with FdI, showed that the alternative salt bridge and the absence of an otherwise conserved Tyr residue are important for the alternative stabilization of the FdII [2Fe-2S] cluster. We also investigated FdI and FdII electron transfer to FNR on chloroplast thylakoid membranes. The K(m) and V(max) values of FdII are similar to those of FdI, contrary to previous measurements of the reverse reaction, from FNR to Fd. The affinity between reduced FdI and oxidized FNR is much greater than that between oxidized FdI and reduced FNR, whereas this is not the case with FdII. The pH dependence of electron transfer by FdI, FdII, and an FdII mutant with FdI features was measured and further indicated that the binding mode to FNR differs between FdI and FdII. Based on this evidence, we hypothesize that binding modes with other Fd-dependent reductases may also vary between FdI and FdII. The structural differences between FdI and FdII therefore result in functional differences that may influence partitioning of electrons into different redox metabolic pathways.     

Interactions between the molybdenum cofactor and iron-sulfur clusters of Escherichia coli dimethylsulfoxide reductase

We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups of Escherichia coli Me2SO reductase (DmsABC). In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = -120 mV [4Fe-4S] cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA. This interaction is significantly modified in a DmsA-C38S mutant that contains a [3Fe-4S] cluster in DmsA, suggesting that the [3Fe-4S] cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = -120 mV cluster of DmsB. In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway. In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized [3Fe-4S] cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum. In a double mutant, DmsAS176ABC102SC, which contains an engineered [3Fe-4S] cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized [3Fe-4S] cluster and the Mo(V). These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the [4Fe-4S] clusters of DmsB to the Mo-bisMGD of DmsA.     

Electron paramagnetic resonance studies of mammalian succinate dehydrogenase. Detection of the tetranuclear cluster S2

Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.     

Crystal structures of ferricyanide-oxidized [Fe-S] clusters in Azotobacter vinelandii ferredoxin I

Crystals of Azotobacter vinelandii ferredoxin I (FdI) have been soaked in solutions containing K₃Fe(CN)₆ in order to study the oxidation of the [3Fe-4S] and [4Fe-4S] clusters in the protein. Ferricyanide treatment results in partial loss of Fe and S from each cluster accompanied by alteration of Fe-S bonds. The effects of oxidation can be quantitated by crystallographic refinement when each [Fe-S] cluster is modeled as having a single, average structure with non-standard geometry. The oxidized clusters refined at 2.1-Å resolution display statistically significant deviations from geometric ideality. If interpreted in terms of atomic shifts these deviations indicate that each cluster first loses an inorganic S atom. In each case an Fe atom bonded to this S separates from the remaining atoms of the cluster such that the [3Fe-4S] and [4Fe-4S] clusters partially decompose into a single Fe plus 2Fe and 3Fe fragments. The extent of structural changes observed are essentially the same in crystals soaked at 3?:?1, 9?:?1 and 30?:?1 mole ratio of K₃ Fe(CN)₆?:?FdI, suggesting that the crystal lattice permits limited oxidation reactions to occur at a low mole ratio but restricts conformational changes from occurring that may be required for more extensive oxidative reactions at higher mole ratio. The results are relevant to understanding the transformations which may take place when [Fe-S] proteins are deliberately oxidized with ferricyanide.     

Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron-sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). In this work, the ground- and excited-state electronic properties of the paramagnetic Fe-S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable-temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe-4S](+) clusters in dithionite-reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe-4S](3+) clusters in oxidized HDR samples treated separately with the CoM-SH and CoB-SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.     

Iron-sulphur clusters in fumarate reductase from Vibrio succinogenes

(1) The fumarate reductase complex from Vibrio succinogenes contains one FAD molecule, one [4Fe-4S]3+(3+,2+) and one [2Fe-2S]2+(2+,1+) cluster per enzyme molecule. Both clusters can be partly reduced by succinate. In the presence of excess Na2S2O4 and fumarate, the [2Fe-2S] cluster is completely oxidized, whereas the other cluster is largely reduced. (2) The [2Fe-2S] cluster is localized in the Mr, 31,000 subunit. The EPR spectrum of the reduced cluster in the isolated subunit differs slightly in line width, but not in g-value, from the spectrum of reduced, intact enzyme complex. The demonstrates that the immediate environment of th cluster is little perturbed by dissociating this subunit from the FAD-containing Mr 79,000 subunit. The temperature dependence of the power-saturation behaviour has, however, greatly decreased in the isolated subunit, the saturation at 11 K of the paramagnetic cluster being much less than in the enzyme complex. Moreover, the temperature dependence of th power-saturation behaviour of this cluster in the enzyme is greater with succinate as reducing agent, than with dithionite. (3) The [4Fe-4S] cluster is located on the Mr 79,000 subunit. This cluster is unstable in air when the subunit has been dissociated from the enzyme complex.     

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy

Low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the metal clusters in the conventional nitrogenase MoFe protein and alternative VFe protein from Azotobacter vinelandii. In the dithionite-reduced state, the MCD spectrum of the MoFe protein is extremely similar to that previously observed for the S = 3/2 spin state of the M clusters in the MoFe protein of Klebsiella pneumoniae. A paramagnetic cluster with an S = 3/2 ground state is also responsible for the temperature-dependent MCD transitions of dithionite-reduced VFe protein. However, the electronic and magnetic properties of this cluster are quite distinct from those of M centers in conventional nitrogenase. When these proteins are oxidized with thionine, the MoFe protein exhibits MCD spectra and magnetization characteristics identical with those observed for the P clusters in K. pneumoniae, while those of the VFe protein are only similar. However, the paramagnetism in the thionine-oxidized VFe protein, like the conventional enzyme, probably arises from an S = 5/2 spin system with near-axial symmetry and a negative zero-field splitting. Novel clusters with electronic, magnetic, and redox properties similar to those of conventional P clusters are, therefore, present in the VFe protein.